Abstract The human kidney is a complex organ reported to contain over 25 cell types spatially organized into a million nephrons connected to a urine-transporting network. However, the actual cellular heterogeneity is likely to be much greater. Together, kidney structures function in heterogeneous environments created by the distinct organization (cortical- medullary) and distribution of distinct cell types. Genetic differences, injury and stress can result in kidney disease. A large gap exists in our understanding of the molecular blueprint that drives homeostatic interactions or pathology in kidney. A complete catalog of molecular signatures of all kidney cell types in 3D space, with single cell resolution, is crucial for an improved understanding of normal kidney functions, and acute and chronic kidney disease (AKD and CKD). In this project we will fill this existing gap with three specific aims. In the UG3 phase, with Aim 1, we will collect, preserve and prepare human kidney specimens from consented patients (existing and new) and optimize a single-nucleus RNA sequencing method to human kidneys. In Aim 2, we will optimize an multiplexed in situ RNA imaging method for spatially mapping the transcripts of 100+ genes within human kidney. In the UH3 phase (Aim 3), we will generate a reference cell atlas of human kidney with cell types, sub-types and their relative composition, a true 3D sub-micron resolution spatial map, and apply this knowledge to uncover molecular differences at a cellular level in CKD